Microbial Synthesis Of Aldehydes And Corresponding Alcohols

ABSTRACT

An improved process for alcohol production includes microbial fermentation using a genetically modified microorganism to produce substantial quantities of aldehydes that are stripped from the fermentation medium and condensed. So produced aldehydes are converted in an ex vivo process to corresponding alcohols.

This application claims priority to our copending U.S. provisionalapplication with the Ser. No. 61/452,519, which was filed Mar. 14, 2011.

FIELD OF THE INVENTION

The field of the invention is metabolic engineering of microorganisms toproduce one or more chemicals, and especially aldehydes, that are thenisolated and converted ex vivo to the corresponding alcohols.

BACKGROUND OF THE INVENTION

World production and consumption of aldehydes and other oxo-chemicalswas nearly 9.6 million metric tons in 2005. Global capacity utilizationincreased to 84% in 2005 from 79% in 2001 as a result of strongerdemand, increased production and rationalized capacity. Between 2001 and2005, world capacity for aldehydes and other oxo-chemicals grew at anaverage annual rate of 1.6%, a lower rate than world consumption, whichgrew at an average annual rate of 3.4% during the same period.

Most commonly, aldehydes and other oxo-chemicals are currently beingproduced by refinery methods using petrochemicals derived from crude oilcracking. For example, C3 to C15 aldehydes are generated viahydroformylation of olefins with synthesis gas, and the so producedaldehydes are then converted to corresponding alcohols, acids, or otherderivatives. Currently, the oxo-chemical in highest demand isn-butyraldehyde, followed by C6-C13 aldehydes for plasticizer alcohols,and isobutyraldehyde and C12-C18 aldehydes for detergent alcohols.

Microbial synthesis of biofuels using metabolically engineered microbialcells, and especially production of C2-C6 alcohols is well known in theart. For example, microbial ethanol production from carbohydrates isdescribed in WO 94/06924 and ethanol production from CO2 is reported inU.S. Pat. No. 8,048,666. Short-chain alcohol production from 2-ketoacids using metabolically engineered cells is described in U.S. Pat.App. No. 2009/0081746, and numerous publications are directed toisobutanol production from metabolically engineered cells (e.g., U.S.Pat. Nos. 7,851,188 and 7,993,889, and in WO 2009/086423, WO2009/149240, WO 2010/062597, and WO 2010/075504), and alcohol productionfrom CO2 using photosynthetically active organisms is described inUS2011/0250660. Similar methods were also described by Kechun Zhang etal. in Proc. Nat. Acad. Sci. (2008), 105, no. 52: 20653-20658. C5-8alcohol production from 2-keto acids using metabolically engineeredcells was described in U.S. Pat. App. No. 2011/0201083, and productionof fatty aldehydes from various carbon sources was reported in U.S. Pat.No. 8,097,439. These and all other extrinsic materials discussed hereinare incorporated by reference in their entirety. Where a definition oruse of a term in an incorporated reference is inconsistent or contraryto the definition of that term provided herein, the definition of thatterm provided herein applies and the definition of that term in thereference does not apply.

Unfortunately, yield of alcohol using many of such processes is stillrelatively low. To improve yield of at least certain alcohols,endogenous alcohol dehydrogenases can be deleted or suppressed, and canbe replaced with a recombinant dehydrogenase as described in WO2009/149240A1. While such modifications are often desirable to at leastsome extent, other problems arise. For example, various alcohols aretoxic to the cells producing the alcohol above a thresholdconcentration, which tends to limit the overall yield. Moreover, mostmicrobially synthesized alcohols are completely miscible with thefermentation medium and need a rather energy consuming process forisolation. Worse, yet, some of the alcohols for azeotropic mixtures andare even more difficult to separate from the medium.

Thus, even though numerous systems and methods of production ofaldehydes, oxo-chemicals, and corresponding alcohols are known in theart, several difficulties nevertheless remain. Therefore, there is stilla need for improvement, particularly where such chemicals are producedusing a microbial system.

SUMMARY OF THE INVENTION

The present invention is directed to devices and methods for productionof aldehyde and alcohol compounds using a mixed synthetic process inwhich a metabolically engineered microbial cell uses a carbon source toproduce an aldehyde that is then continuously or semi-continuouslyremoved in the vapor phase from the fermentation medium. In particularlypreferred aspects, the metabolically engineered microbial cell issubstantially devoid of any alcohol production. The aldehyde is thencondensed from the vapor phase and reduced ex vivo to the correspondingalcohol. Contemplated methods advantageously overcome variousdifficulties, especially various problems associated with productinhibition and separation of the alcohols from the fermentation medium.

In one aspect of the inventive subject matter a method of producing analcohol includes a step of growing a plurality of microbial cells in afermentation medium (preferably having glucose, fructose, sucrose,starch, cellulose, a hemicellulose, glycerol, carbon dioxide, a protein,a lipid, and/or an amino acid as carbon source), wherein the cells aregenetically modified to have an increased metabolic activity as comparedto non-genetically modified cells. It is especially preferred that theincreased metabolic activity is an increased conversion of pyruvate or2-ketobutyrate to an aldehyde, and a decreased alcohol dehydrogenaseactivity. Aldehyde produced by the cells is continuously orsemi-continuously removed from the fermentation medium in the vaporphase. In another step, the aldehyde is condensed from the vapor phase,and in yet another step the condensed aldehyde is reduced to thecorresponding alcohol.

While in some embodiments the increased metabolic activity is anincreased conversion of pyruvate to acetaldehyde via a recombinantpyruvate decarboxylase, the increased metabolic activity in otherembodiments is an increased conversion of 2-ketobutyrate to propanal viaa recombinant 2-ketoisovalerate decarboxylase. Additionally, oralternatively, it is preferred that the microbial cells also haveincreased metabolic activity in decarboxylation of one or more of2-ketovalerate, 2-ketocaproate, 2-ketoheptanoate, 2-ketooctanoate,2-keto-3-methylvalerate, 2-keto-4-methylcaproate,2-keto-5-methylheptanoate, 2-keto-6-methyloctanoate, 2-keto-isovalerate,2-ketoisocaproate, 2-keto-5-methylhexanoate, or 2-keto-6-methylocatnoatevia a recombinant 2-ketoisovalerate decarboxylase. Thus, especiallypreferred fermentation produces include acetaldehyde, propanal, butanal,and 2-methyl-1-propanal.

In still further contemplated aspects, the microbial cells haveincreased metabolic activity in branched carbon chain elongation of2-ketobutyrate to 2-keto-3-methylvalerate via recombinant ilvGMCD genesor recombinant ilvBNCD genes, and/or increased metabolic activity inbranched carbon chain elongation of pyruvate to 2-keto-isovalerate viarecombinant alsS-ilvCD genes or recombinant ilvIHCD genes. It is stillfurther particularly preferred that the microbial cells also haveincreased metabolic activity in linear carbon chain elongation viarecombinant leuABCD genes.

While not limiting to the inventive subject matter, it is generallypreferred that the decreased alcohol dehydrogenase activity in themicrobial cells is decreased at least 70% and more typically at least90% as compared to the non-genetically modified cells.

In particularly preferred methods, the step of removing the aldehyde inthe vapor phase includes agitation of the fermentation medium, strippingthe fermentation medium with an inert gas, stripping the fermentationmedium with an oxygen containing gas, and/or temporarily binding thealdehyde to a binding agent. Most typically, the aldehyde iscontinuously removed from the fermentation medium. The so isolatedaldehyde is then reduced to the corresponding alcohol, for example,using electrochemical reduction, enzymatic reduction, and/or a catalyticreduction with hydrogen.

Suitable microbial cells will be selected from the genera Escherichia,Bacillus, Corynebacterium, Ralstonia, Zymomonas, Clostridium,Lactobacillus, Synechococcus, Synechocystis, Saccharomyces, Pichia,Candida, Hansenula, and Aspergillus. Thus, particularly preferredmicrobial cells include Escherichia coli, Bacillus subtilis,Synechococcus elongatus, Ralstonia eutropha, and Saccharomycescerevisiae.

Therefore, and viewed from a different perspective, a method ofproducing a metabolically engineered microbial cell for use in anproduction process in which a value product (e.g., alcohol) is ex vivoproduced from an aldehyde will include a step of genetically modifyingthe microbial cells to have an increased conversion of pyruvate or2-ketobutyrate to an aldehyde, and a further step of geneticallymodifying the microbial cells to have a decreased alcohol dehydrogenaseactivity such that the microbial cell is substantially devoid of alcoholproduction. In yet another step, the modified microbial cells are testedfor generation in a fermentation medium of a volatile aldehyde in aquantity sufficient to allow stripping of the volatile aldehyde from thefermentation medium to thereby allow for an ex vivo conversion of thealdehyde to the value product (e.g., reduction of the aldehyde to acorresponding alcohol).

In especially preferred aspects, the microbial cell has increasedmetabolic activity in conversion of pyruvate to acetaldehyde via arecombinant pyruvate decarboxylase or an increased metabolic activity inconversion of 2-ketobutyrate to propanal via a recombinant2-ketoisovalerate decarboxylase. Additionally, or alternatively, themicrobial cell has an increased metabolic activity in linear carbonchain elongation via recombinant leuABCD genes, and/or an increasedmetabolic activity in branched carbon chain elongation of 2-ketobutyrateto 2-keto-3-methylvalerate via recombinant ilvGMCD genes or recombinantilvBNCD genes or in branched carbon chain elongation of pyruvate to2-keto-isovalerate via recombinant alsS-ilvCD genes or recombinantilvIHCD genes.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is an exemplary schematic for metabolic pathways that producelinear chain aldehydes.

FIG. 1B is an exemplary schematic for metabolic pathways that producebranched chain aldehydes.

DETAILED DESCRIPTION

The inventors have discovered that microbial cells can be metabolicallyengineered to substantially increase production of various (especiallyvolatile) aldehydes that are not or only to a negligible degree furthermetabolized to the corresponding alcohols. Such approach is particularlyunexpected as aldehydes are typically significantly more toxic than thecorresponding alcohols, and as the aldehydes will be produced (andpotentially accumulated in the cell) at an even faster rate due to thesuppression of the endogenous alcohol dehydrogenases. The so producedaldehydes are then removed from the fermentation medium in the vaporphase, preferably by stripping the medium with a stripping gas in acontinuous or semi-continuous manner (i.e., in an intermittent fashionthroughout the fermentation process using at least two removal periods).

In especially preferred aspects of the inventive subject matter, themicrobial cell is genetically modified to have an increased conversionof pyruvate or 2-ketobutyrate to an aldehyde, and a decreased alcoholdehydrogenase activity. Increased conversion of pyruvate or2-ketobutyrate to the aldehyde is most typically due to the presence ofone or more nucleic acid constructs (e.g., provided as plasmids and/orintegrated into the host cell genome) that encode one or more genes thatlead to the formation of enzymes that catalyze a reaction in theconversion of pyruvate or 2-ketobutyrate to the aldehyde. Thus, in mostcases, the increased conversion is due to a higher throughput ofmetabolites through a sequence of biochemical reactions in the cell thatlead to the desired aldehyde end product(s). Of course, it should beappreciated that one or more endogenous (non-recombinant) enzymes may bepart of the sequence of biochemical reactions in the cell.

FIG. 1A depicts a set of exemplary metabolically engineered pathways forincreased production of linear chain aldehydes in a cell. Here,acetaldehyde is formed from pyruvate via the enzyme pyruvatedecarboxylase (pdc), and propanal is formed from 2-ketobutyrate (2-KB)via the enzyme 2-ketoisovalerate decarboxylase (kivD). To arrive atlonger-chain products, including butanal, pentanal, hexanal, heptanal,etc., metabolically engineered pathways may further include the genesencoding an 2-isopropylmalate synthase (leuA), an 3-isopropylmalatedehydrogenase (leuB), a 3-isopropylmalate isomerase large subunit(leuC), and a 3-isopropylmalate isomerase, small subunit (leuD). Mostpreferably, these genes are arranged in an operon under appropriatecontrol for expression in a cell. Thus, cells engineered to expressleuABCD and kivD will be suitable for production of butanal, pentanal,hexanal, heptanal, etc. from 2-ketobutyrate as depicted throughsuccessive chain extension from 2-KB to 2-ketovalerate (2-KV),2-ketocaproate (2-KC), 2-ketoheptanoate (2-KH), and 2-ketooctanoate(2-KO) and final decarboxylation.

FIG. 1B depicts another set of exemplary metabolically engineeredpathways for increased production of branched chain aldehydes in a cell.Here, 2-KB is branched to for 2-Ket-3-methylvalerate (2-K-3-MV) viaaction of gene products of the large subunit of acetohydroxy acidsynthase II (ilvG), the small subunit of acetohydroxy acid synthase II(ilvM), acetohydroxy acid isomeroreductase (ilvC), and dihydroxy aciddehydratases (ilvD) or the large subunit of acetohydroxy acid synthase I(ilvB), the small subunit of acetohydroxy acid synthase I (ilvN),acetohydroxy acid isomeroreductase (ilvC), and dihydroxy aciddehydratase (ilvD). 2-K-3-MV may then either the decarboxylated by kivDto form 2-methyl-1-butanal, or may be successively elongated viaproteins encoded by leuABCD to 2-keto-4-methylhexanoate (2-K-4 MH) or2-keto-5-methylheptanoate (2-K-5 MHp) prior to decarboxylation by kivDto form respective 3-methyl-1-pentanal and 4-methyl-1-hexanal (andlonger branched products). Where the starting material is pyruvate, thepyruvate is first branched by the expression products of theacetolactate synthase (alsS), acetohydroxy acid isomeroreductase (ilvC),and dihydroxy acid dehydratase (ilvD) genes, preferably arranged in afunctional expression cassette alsS-ilvCD, or the expression products ofthe large subunit of acetohydroxy acid synthase III (ilvI), the smallsubunit of acetohydroxy acid synthase III (ilvH), acetohydroxy acidisomeroreductase (ilvC), and dihydroxy acid dehydratase (ilvD) genes toform 2-ketoisovalerate (2-KIV). Decarboxylation of 2-KIV yields2-methyl-1-propanal, while chain elongation via expression products ofleuABCD yields 2-ketoisocaproate (2-KIC) and 2-keto-5-methylhexanoate(2-K-5 Mhx) and higher products. As before, 2-KIC and 2-K-5 Mhx are thendecarboxylated to the corresponding 3-methyl-1-butanal and4-methyl-1-pentanal, and higher products.

Therefore, in especially preferred aspects of the inventive subjectmatter, microbial cells contemplated herein will have increasedmetabolic activity in branched carbon chain elongation of 2-ketobutyrateto 2-keto-3-methylvalerate via expression of recombinant ilvGMCD genesor expression of recombinant ilvBNCD genes, and/or increased metabolicactivity in branched carbon chain elongation of pyruvate to2-keto-isovalerate via expression of recombinant alsS-ilvCD genes orexpression of recombinant ilvIHCD genes.

In still further preferred aspects, contemplated cells will also have anincreased metabolic activity in decarboxylation of one or more of2-ketovalerate, 2-ketocaproate, 2-ketoheptanoate, 2-ketooctanoate,2-keto-3-methylvalerate, 2-keto-4-methylcaproate,2-keto-5-methylheptanoate, 2-keto-6-methyloctanoate, 2-keto-isovalerate,2-ketoisocaproate, 2-keto-5-methylhexanoate, or 2-keto-6-methylocatnoatevia expression of a recombinant 2-ketoisovalerate decarboxylase(preferably kivD), and/or an increased metabolic activity in conversionof pyruvate to acetaldehyde via expression of a recombinant pyruvatedecarboxylase, and/or an increased metabolic activity in conversion of2-ketobutyrate to propanal via expression of a recombinant2-ketoisovalerate decarboxylase. In particularly preferred aspects,cells will further be genetically modified to have an increasedmetabolic activity in linear carbon chain elongation via expression ofrecombinant leuABCD genes.

Of course, it should be recognized that all of the genes may beunmodified or may be engineered to impart a desired selectivity, anincreased turnover rate, etc. (see e.g., Proc. Nat. Acad. Sci. (2008),105, no. 52: 20653-20658; WO2009/149240A1). Suitable genes for theactivities of the metabolically engineered cells are well known in theart, and use of all of those in conjunction with the teachings presentedherein is deemed suitable. Moreover, all of the known manners of makingmetabolically engineered cells are also deemed suitable for use herein.For example, metabolically engineered cells may modified by genomicinsertion of one or more genes, operons, or transfection with plasmidsor phagemids as is well known in the art. In some embodiments, a mutantmicroorganism may also be used in the methods of the present invention,and may be further modified recombinantly as desired.

In further particularly preferred aspects, endogenous alcoholdehydrogenase activity is at least decreased, and more preferablysuppressed, and it should be noted that in preferred aspects, all oralmost all of the alcohol dehydrogenases will be suppressed or deleted.For example, suppressed or deleted dehydrogenases include adhE, IdhA,frdB, and pflB. It is also noted that dehydrogenase activity can besuppressed or deleted suppressed in numerous well known manners,including down-regulation (e.g., via antisense RNA or siRNA) ordisruption of a gene encoding the dehydrogenase, introduction of aknock-down or knock-out mutation, etc.). Consequently, contemplatedgenetically modified cells will have more than one dehydrogenase mutatedor otherwise suppressed.

Viewed from a different perspective, it is therefore contemplated thatthe genetically modified cells will not produce any significantquantities of short-chain (up to C6, linear or branched) alcohols. Forexample, such modified cells will release into the fermentation mediasignificantly higher quantities of aldehydes relative to thecorresponding alcohols, most typically at a molar ratio of an aldehydeto a corresponding alcohol (e.g., butyraldehyde to butanol) of at least3:1, more typically at least 4:1, and most typically at least 5:1.Consequently, total short-chain (up to C6, linear or branched) alcoholin the fermentation medium will be less than 1 wt % of the fermentationmedium, more typically less than 0.5 wt %, most typically less than 0.1wt %. Thus, in especially preferred aspects, modified cells will notproduce any detectable alcohol (i.e., less than 10 mg/l fermentationmedium).

The recombinant microorganism may be any suitable microorganism,including bacteria, cyanobacteria, or a fungus. However,non-photosynthetically active microorganisms are particularly preferred.Therefore, in some embodiments, the microbial cells belong to a genusselected from the group consisting of Escherichia, Bacillus,Corynebacterium, Ralstonia, Zymomonas, Clostridium, Lactobacillus,Synechococcus, Synechocystis, Saccharomyces, Pichia, Candida, Hansenula,and Aspergillus. In preferred embodiments, the microorganism isconsisting Escherichia coli, Bacillus subtilis, Synechococcus elongatus,Ralstonia eutropha, and Saccharomyces cerevisiae.

It should further be appreciated that the culture conditions willtypically depend on the particular choice of microorganism, and theperson of ordinary skill in the art will be readily able to chose theappropriate medium. Among other suitable choices, it is generallypreferred that the carbon source in the medium is a saccharide, andparticularly glucose, fructose, sucrose, starch, cellulose, ahemicellulose, glycerol, carbon dioxide, a protein, and/or an aminoacid. However, numerous alternative carbon sources are also deemedsuitable, and exemplary further carbon sources include lipids, proteins,CO2, CH4, complex organic mixtures (e.g., biosolids, meat processingwaste products, plant based materials, etc.) Regardless of theparticular culture condition, the volatile aldehyde is removed from thefermentation medium in the vapor phase. More preferably, such removalwill be performed in a continuous fashion during cell culture, andremoval may be based on agitation of the fermentation medium, strippingthe fermentation medium with an inert gas, stripping the fermentationmedium with an oxygen containing gas, and/or temporarily binding thealdehyde to a binding agent. Alternatively, aldehyde removal may also beperformed after fermentation, or in a semi-continuous manner (e.g., byintermittent contact with stripping gas).

With respect to further processing, it should be recognized thatcondensation of the aldehyde may be performed in various manners,preferably using a condenser well known in the art, and that the socondensed aldehyde product may be further purified in one or more stepsusing conventional manners, or may be directly used in a reductionreaction to produce the corresponding alcohol. There are numerousreduction reactions for aldehydes known in the art, and all of them aredeemed suitable for use herein. For example, especially suitablereduction reactions include electrochemical reduction, an enzymaticreduction, and a catalytic reduction with hydrogen.

Therefore, it should be appreciated that a method of producing ametabolically engineered microbial cell for use in an alcohol productionprocess will include genetically modifying the microbial cells to havean increased conversion of pyruvate or 2-ketobutyrate to an aldehyde;genetically modifying the microbial cells to have a decreased alcoholdehydrogenase activity such that the microbial cell is substantiallydevoid of alcohol production; and testing the modified microbial cellsfor generation in a fermentation medium of a volatile aldehyde in aquantity sufficient to allow stripping of the volatile aldehyde from thefermentation medium to thereby allow for an ex vivo reduction of thealdehyde to a corresponding alcohol.

Where desired, it is also contemplated that the cells presented hereinneed not be employed for alcohol production, but for generation ofvarious aldehydes, and especially volatile aldehydes. In such case, thecells are genetically modified as described above and cultivated underconditions suitable for production of the aldehyde. Most typically, theso produced aldehyde is then stripped from the culture medium andcondensed for sale, or further use or storage.

Additionally, it should be appreciated that contemplated methods, cells,and processes will also advantageously allow production of desirablecompounds other that alcohols where a precursor of the desirablecompound is an aldehyde. For example, where the fermentation product ofthe genetically modified cell is acetaldehyde, especially preferredproduct that are prepared ex vivo from acetaldehyde are ethanol andacetic acid. Similarly, where the fermentation product of thegenetically modified cell is propionaldehyde, especially preferredproduct that are prepared ex vivo from propionaldehyde includen-propanol, n-propyl acetate, propionic acid, and cellulose acetatepropionate. Likewise, where the fermentation product of the geneticallymodified cell is n-butyraldehyde, especially preferred product that areprepared ex vivo from n-butyraldehyde include n-Butanol, 2-ethylhexanol,poly vinyl butyral, 2-ethylhexanoic acid, methyl amyl ketone, n-butyricacid, n-butyl acetate, n-butyl acrylate, and cellulose acetate butyrate.Where the fermentation product of the genetically modified cell isisobutyraldehyde, especially preferred product that are prepared ex vivofrom isobutyraldehyde include isobutanol, isobutyric acid, neopentylglycol, methyl isoamyl ketone, isobutyl acetate, isobutyl acrylate,2,2,4-trimethyl-1,3-pentanediol, 2,2,4-trimethyl-1,3-pentanediol,monoisobutyrate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, andisobutyl isobutyrate.

EXAMPLES

DNA Manipulation: Standard recombinant DNA technologies were used in theexamples, which are well known to the skilled man in this field asdescribed in Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1989) by Sambrook J et al.

Gene Knockout: All the genetic knock-outs were achieved with P1transduction using appropriate Keio collection strains (Construction ofEscherichia coli K-12 in-frame, single-gene knockout mutants: the Keiocollection, Mol. Syst. Biol. 2:2006.0008 (2006)). The Kanamycinresistance gene were eliminated using pCP20 (One-step inactivation ofchromosomal genes in E. coli using PCR products, Proc. Natl. Acad. Sci.,97: 6640-6645 (2000)) in between each consecutive knockout.

Fermentation: E. coli strains were cultured overnight in LB withappropriate antibiotics at 37° C. The next day, the overnight cells weresubcultured (usually 1:100) in 250 ml-screw cap flasks containing 10-20ml of M9 media (64 g Na2HPO4.7H2O, 15 g KH2PO4, 2.5 g NaCl, 5 g NH4Cl, 2mM MgSO4, 0.1 mM CaCl2, 10 mg thiamine per liter water) with 5 g/literyeast extract, 40 g/liter glucose, and appropriate antibiotics. Thecultures were then incubated at 37° C. in a rotary shaker (250 rpm). Toreduce the loss of isobutyraldehyde, the cultures were chilled to 4° C.for 30 min prior to sampling.

GC Analysis: Isobutyraldehyde and isobutanol were quantified by gaschromatography (GC) equipped with a FID (flame ionization detector). Thesystem consisted of a model 7890A GC and model 7693 automatic injector(Agilent Technologies). A 30m with internal diameter of 0.32 mm, 0.25 μmDB-FFAP capillary columns (Agilent Technologies) was used. GC oventemperature was held at 85° C. for 3 min and raised with a gradient of45° C./min until 225° C. and held 3 min. Detector and Inlet temperaturewas held at 225° C. Hydrogen, air and helium gas was used with flowrates of 40 ml/min, 450 ml/min, 45 ml/min, respectively. The supernatantof culture broth was injected in split injection mode (1:25 split ratio)using 1-pentanol as the internal standard.

Construction of the Plasmids for Isobutyraldehyde Production: pEB121:Plasmid pEB0121 was constructed by DNA assembly of four fragments. Thefirst fragment, containing the PLlacO1 promoter, p15A replicationorigin, and the gene for kanamycin resistance, was amplified withprimers 1 and 2 from a derivative of pZE21-MCS1 (Independent and tightregulation of transcriptional units in Escherichia coli via the LacR/O,the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res25:1203-10 (1997)). In this pZE21-MCS1 derivative, PLtetO1 is replacedwith PLlacO1 as a result of a promoter swap with pZE12-luc (Independentand tight regulation of transcriptional units in Escherichia coli viathe LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic AcidsRes 25:1203-10 (1997)) using the AatI and Acc65I restriction sites. Thesecond fragment was amplified with primers 3 and 4 from B. subtilisgenomic DNA as the template. The third fragment contained ilvC, whichwas amplified with primers 5 and 6 from E. coli genomic DNA (ATCC10789D-5) as the template. The fourth fragment containing ilvD wasamplified with primers 7 and 8 with E. coli genomic DNA (ATCC 10789D-5)as the template.

Primer 1: 5′- ACGCGTGCTAGAGGCATCAAA-3′; Primer 2:5′- TGTACCTTTCTCCTCTTTAATGAATTCGGTCAGTGCG -3′; Primer 3:5′- TTAAAGAGGAGAAAGGTACAATGTTGACAAAAGCAACAAAAGAAC AAA -3′; Primer 4:5′- CATGGTGATTCCTCGTCGACCTAGAGAGCTTTCGTTTTCA -3′; Primer 5:5′- GTCGACGAGGAATCACCATGGCTAACTACTTCAATAC -3′; Primer 6:5′- ATGGTATATCTCCTTCCGGGTTAACCCGCAACAGCAATAC -3′; Primer 7:5′- CCCGGAAGGAGATATACCATGCCTAAGTACCGTTCCGC -3′; Primer 8:5′- TTGATGCCTCTAGCACGCGTTTAACCCCCCAGTTTCGATT -3′.

pEB5: Plasmid pEB0005 was constructed by DNA assembly of two fragments.The vector was amplified by amplification of pZE12-luc (Independent andtight regulation of transcriptional units in Escherichia coli via theLacR/O, the TetR/O and AraC/1142 regulatory elements. Nucleic Acids Res25:1203-10 (1997)) with primers 9 and 10. The kivd gene was amplifiedfrom Lactococcus lactis genomic DNA with primers 11 and 12.

Primer 9: 5′- TCTAGAGGCATCAAATAAAACGAAAGG -3′; Primer 10:5′- GGTACCTTTCTCCTCTTTAATGAATTC -3′; Primer 11:5′- TTAAAGAGGAGAAAGGTACCATGTATACAGTAGGAGATTA -3′; Primer 12:5′- TTTTATTTGATGCCTCTAGAATGATTTATTTTGTTCAGCA -3′

Construction of the Isobutyraldehyde Production Host Strain: E. coliBW25113 (Datsenko and Warner 2000) was used as wild-type. To eliminateIPTG induction, lad gene was first knocked out. And then, the majoralcohol dehydrogenases are serially knocked out to construct platformstrain, EB4 (E. coli BW25113ΔlacIΔadhEΔyqhDΔyiaY), for isobutyraldhydeproduction. Elimination of alcohol dehydrogenases could enhanceisobutyraldehyde production by blocking conversion into isobutanol asshown in Table 1 (All strains are harboring two plasmids, pEB5 and pEB121).

TABLE 1 Isobutyraldehyde Isobutanol Strain (g/L) (g/L) BW25113 1.4 5.5BW25113ΔlacI 1.5 5.5 BW25113ΔlacIΔadhE 3.5 3.0 BW25113ΔlacIΔadhEΔyqhD4.5 1.2 BW25113ΔlacIΔadhEΔyqhDΔyiaY 5.0 0.5

Elimination of Residual Alcohol Dehydrogenase Activity enhancedIsobutyraldehyde Production: Microorganisms have lots of non-specificalcohol dehydrogenase genes. Especially, in E. coli, more than 100alcohol dehydrogenases were found by searching enzyme data bases.Eliminating those non-specific alcohol dehydrogenases from EB4 strainwould be beneficial for the isobutyraldehyde production by preventingconversion into isobutanol. In this example, we further eliminated thecandidate alcohol dehydrogenase gene, yjgB, to check this hypothesis. Asshown in Table 2, further elimination of residual alcohol dehydrogenaseactivity was beneficial for the isobutyraldehyde production.

TABLE 2 Isobutyraldehyde (g/L) Isobutanol (g/L) EB4 5.49 3.9 (pEB5 +pEB121) EB4ΔyjgB 6.25 2.61 (pEB5 + pEB121)

Knockout for Competing Metabolic Pathways to enhance IsobutyraldehydeProduction: Metabolic pathway for isobutyraldehyde production hasseveral competing pathways for the utilization of key intermediatesincluding pyruvate and 2-ketoisovalerate. By knocking out thosecompeting pathway genes, we could enhance metabolic carbon flow towardour isobutyraldehyde production pathway. To do this, we selected poxB,which convert pyruvate to acetate and ilvE, which convert2-ketoisovalerate to L-valine. As shown Table 3, it was found to enhanceisobutyraldehyde production slightly. However, by combining thosecompeting pathway knockouts, the effect on isobutyraldehyde productionwas significant as shown in Table 4.

TABLE 3 Isobutyraldehyde (g/L) Isobutanol (g/L) EB4 5.45 1.5 (pEB5 +pEB121) EB4ΔilvE 5.55 1.4 (pEB5 + pEB121) EB4ΔpoxB 5.77 1.5 (pEB5 +pEB121)

TABLE 4 Isobutyraldehyde (g/L) Isobutanol (g/L) EB4 7.40 1.7 (pEB5 +pEB121) EB4ΔilvEΔpoxB 8.30 2.0 (pEB5 + pEB121)

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of producing an alcohol, comprising:growing a plurality of microbial cells in a fermentation medium, whereinthe cells are genetically modified to have an increased metabolicactivity as compared to non-genetically modified cells; wherein theincreased metabolic activity is characterized by increased conversion ofpyruvate or 2-ketobutyrate to an aldehyde; wherein the plurality ofmicrobial cells are further genetically modified to have a decreasedalcohol dehydrogenase activity; continuously or semi-continuouslyremoving the aldehyde in a vapor phase from the fermentation medium;condensing the aldehyde from the vapor phase; and reducing the condensedaldehyde to the corresponding alcohol.
 2. The method of claim 1 whereinthe microbial cells have increased metabolic activity in conversion ofpyruvate to acetaldehyde via expression of a recombinant pyruvatedecarboxylase.
 3. The method of claim 1 wherein the microbial cells haveincreased metabolic activity in conversion of 2-ketobutyrate to propanalvia expression of a recombinant 2-ketoisovalerate decarboxylase.
 4. Themethod of claim 1 wherein the microbial cells have increased metabolicactivity in decarboxylation of one or more of 2-ketovalerate,2-ketocaproate, 2-ketoheptanoate, 2-ketooctanoate,2-keto-3-methylvalerate, 2-keto-4-methylcaproate,2-keto-5-methylheptanoate, 2-keto-6-methyloctanoate, 2-keto-isovalerate,2-ketoisocaproate, 2-keto-5-methylhexanoate, or 2-keto-6-methylocatnoatevia expression of a recombinant 2-ketoisovalerate decarboxylase.
 5. Themethod of claim 1 wherein the microbial cells have increased metabolicactivity in branched carbon chain elongation of 2-ketobutyrate to2-keto-3-methylvalerate via expression of recombinant ilvGMCD genes orexpression of recombinant ilvBNCD genes.
 6. The method of claim 1wherein the microbial cells have increased metabolic activity inbranched carbon chain elongation of pyruvate to 2-keto-isovalerate viaexpression of recombinant alsS-ilvCD genes or expression of recombinantilvIHCD genes.
 7. The method of any one of claim 4, claim 5, or claim 6,wherein the microbial cells have increased metabolic activity in linearcarbon chain elongation via expression of recombinant leuABCD genes. 8.The method of claim 1 wherein the decreased alcohol dehydrogenaseactivity in the microbial cells is decreased at least 70% as compared tothe non-genetically modified cells.
 9. The method of claim 1 wherein thedecreased alcohol dehydrogenase activity in the microbial cells isdecreased at least 90% as compared to the non-genetically modifiedcells.
 10. The method of claim 1 wherein the step of removing thealdehyde in the vapor phase comprises a step selected from the groupconsisting of agitation of the fermentation medium, stripping thefermentation medium with an inert gas, stripping the fermentation mediumwith an oxygen containing gas, and temporarily binding the aldehyde to abinding agent.
 11. The method of claim 1 wherein the step of removingthe aldehyde is continuously performed.
 12. The method of claim 1wherein the aldehyde is selected form the group consisting ofacetaldehyde, propanal, butanal, and 2-methyl-1-propanal.
 13. The methodof claim 1 wherein the step of reducing the condensed aldehyde to thecorresponding alcohol is selected from the group of an electrochemicalreduction, an enzymatic reduction, and a catalytic reduction withhydrogen.
 14. The method of claim 1 wherein the microbial cells belongto a genus selected from the group consisting of Escherichia, Bacillus,Corynebacterium, Ralstonia, Zymomonas, Clostridium, Lactobacillus,Synechococcus, Synechocystis, Saccharomyces, Pichia, Candida, Hansenula,and Aspergillus.
 15. The method of claim 14 wherein the microbial cellsbelong to a species selected from the group consisting Escherichia coli,Bacillus subtilis, Synechococcus elongatus, Ralstonia eutropha, andSaccharomyces cerevisiae.
 16. The method of claim 1 wherein thefermentation medium has a carbon source selected form the groupconsisting of glucose, fructose, sucrose, starch, cellulose, ahemicellulose, glycerol, carbon dioxide, a protein, and an amino acid.17. A method of producing a metabolically engineered microbial cell foruse in a process in which a value product is ex vivo produced from analdehyde is, comprising: genetically modifying the microbial cells tohave an increased conversion of pyruvate or 2-ketobutyrate to analdehyde; genetically modifying the microbial cells to have a decreasedalcohol dehydrogenase activity such that the microbial cell issubstantially devoid of alcohol production; and testing the modifiedmicrobial cells for generation in a fermentation medium of a volatilealdehyde in a quantity sufficient to allow stripping of the volatilealdehyde from the fermentation medium to thereby allow for an ex vivoconversion of the aldehyde to the value product.
 18. The method of claim17 wherein microbial cell has increased metabolic activity in conversionof pyruvate to acetaldehyde via expression of a recombinant pyruvatedecarboxylase or an increased metabolic activity in conversion of2-ketobutyrate to propanal via expression of a recombinant2-ketoisovalerate decarboxylase.
 19. The method of claim 17 whereinmicrobial cell has an increased metabolic activity in linear carbonchain elongation via expression of recombinant leuABCD genes.
 20. Themethod of claim 17 wherein microbial cell has an increased metabolicactivity in branched carbon chain elongation of 2-ketobutyrate to2-keto-3-methylvalerate via expression of recombinant ilvGMCD genes orexpression of recombinant ilvBNCD genes, or in branched carbon chainelongation of pyruvate to 2-keto-isovalerate via expression ofrecombinant alsS-ilvCD genes or expression of recombinant ilvIHCD genes.